Spectrophotometric Fluoride Determination Using St. John’s Wort Extract as a Green Chromogenic Complexant for Al(III)

In this study, we applied an innovative approach of green analytical chemistry to develop a novel and eco-friendly chromogenic agent for fluoride determination by making use of the nontoxic Al(III)-flavonoid complex in a natural extract from St. John’s wort plant. The initial intensely yellow-colored Al(III)-flavonoid complex formed in the plant extract was converted to a colorless AlF63– complex with increasing amounts of fluoride, and color bleaching of the Al-flavonoid chromophore (measured as absorbance decrement) was proportional to fluoride concentration. The developed method gave a linear response within the F– concentration range of 0.11–1.32 mM with the LOD and LOQ values of 0.026 mM (0.5 mg L–1) and 0.079 mM (1.5 mg L–1), respectively. The LOD value for fluoride was below the WHO-permissible limit (1.5 mg L–1) and the US-EPA-enforceable limit (4 mg L–1) in water. The possible interference effects of common anions (Cl–, Br–, I–, NO3–, HCO3–, SO42–, and PO43–) and cations (K+, NH4+, Ag+, Ca2+, Mg2+, Mn2+, Fe2+, and Fe3+) were investigated; the observed interferences from Fe2+, Fe3+, and PO43– were easily eliminated by masking iron with the necessary amount of Na2EDTA without affecting the blank absorbance of the Al(III)-flavonoid complex, precipitating phosphate with Ag(I) salt, and partly neutralizing alkaline water samples to pH 4 with acetic acid. The developed method was applied to real water samples and also validated against a reference spectroscopic method at the 95% confidence level.


INTRODUCTION
Healthy and safe water does not contain disease-causing microorganisms and toxic substances but instead contains macro-, micro-, and trace minerals, which are necessary for the human body in a balanced way. Water and health are directly related to each other. One of the important parameters of water quality is the amount of fluoride. Fluorine is the 13th most abundant element in the world and cannot be freely found in the environment unless it combines with other substances to form fluoride. Fluorides can be classified such as ionizable/non-ionizable and organic/inorganic. Organic fluorides do not dissolve as rapidly in water as inorganic fluoride ions not having a chemical reaction with the solvent. 1 Fluoride, as a trace ion in water, is necessary for the growth and development of some organs, especially teeth and bones, when taken into the body at appropriate concentrations (in the range of 0.7−1.2 mg L −1 ). 2 Industrial sectors such as the aluminum industry, oil refineries, steel production, coal processing plants, glass processing, ceramic factories, brickworks and phosphate fertilizer production, and pesticides in agricultural activities are responsible for the excess amount of human-related fluoride contamination. 3 Consumption of fluoride in high concentrations causes dental and skeletal fluorosis, 4 parathyroid gland damage, 5 neurological disorders, 6 and cardiovascular problems. 7 Determination of fluoride concentration has been one of the important issues researched by analytical chemists because of the concentration range limitation in drinking water in terms of human health. 8 The traditional analytical methods for the determination of fluoride ions in water are ion chromatography (IC), 9 gas chromatography (GC), 10 ICP-MS, 11 AAS (i.e., by depression of Mg absorption), 12 and ion-selective electrode-based potentiometry (ISE). 13,14 Among these methods of F − determination, ISE and spectrophotometry stand out to give satisfactory results. 15 The disadvantages of the ISE method were reported as limited selectivity, poor precision, long equilibration time, electrode drifting, and low solubility of the lanthanum fluoride membrane crystal 16−19 when determining fluoride at low concentrations. In addition, the ion activity, presence of interfering ions and colloidal particles, and color and temperature of solution medium may cause problems on the ISE method. 20 Chromatographic methods for the determination of fluoride are expensive and time-consuming and require skilled specialists. 21 Spectrophotometry is a versatile and good alternative technique for determining the concentration of inorganic ions in water samples, having many advantages such as low cost, ease of applicability, fast analysis, reliability, high sensitivity at low concentrations, and wide analytical working range. 22 In spectrophotometric methods, dyestuff-metal complexes, 23−26 nanoparticles, 27,28 or synthesized organic molecules 29,30 have been studied as the three kinds of analytical probes. However, it is known that lots of dyestuffs are both toxic and carcinogenic. 31 Most dyes, especially synthetic ones, are nondegradable due to their stability to light and oxidants. 32 On the other hand, considering the nanoparticle-based syntheses, some of the reducing agents used to synthesize nanoparticles may be toxic, high-priced, of low reducing capability, and may bear a contamination risk (as they can bring other impurities to the system). 33 The synthesis of organic molecules as a fluoride receptor requires both a long time and organic solvents, limiting the use of these analysis systems in aqueous solutions. 34 Organic solvents cause formation of hazardous wastes and also constitute a major source of volatile organic compound (VOC) emission, threatening human health. 35,36 The US-EPA declared that to overcome the problem of disposal of hazardous wastes used in academic studies, attention should be given to the reduction of hazardous wastes. 37 The usage of naturally sourced reagents like plant compounds instead of synthetic toxic chemicals is one of the ways to apply sustainable development principles in analytical laboratories. 38 Lately, it has become popular to approach green analytical chemistry by enhancing currently applied methods and/or developing new methods using eco-friendly materials. 39 Flavonoids are plant-derived polyphenolic compounds having many favorable biochemical properties. 40 St. John's wort (Hypericum perforatum L.) is one of the flavonoid-rich plants 41 and is conventionally consumed as a herbal tea and nutritional supplement due to its remarkable bioactive properties. 42 Flavonoids have the ability to form colored complexes with metal ions due to their carbonyl and hydroxyl groups arranged in a special (usually chelating) geometry. Metal ion coordination by flavonoids causes significant differences in certain properties, e.g., color, fluorescence, oxidation state, catalytic ability, stability, and toxicity, explaining the wide use of flavonoids in analytical chemistry, photochemistry, medicinal chemistry, and textile dyeing. 40,43 In addition, a method based on complexing flavonoids with aluminum(III) is used to find the total flavonoid content of some species. 44 Pekal and Pyrzynska 45 studied and compared two common spectrophotometric procedures, named procedure 1 and procedure 2, to determine the "total flavonoid content" of food and medicinal plant samples. Method 1 involved the measurement of flavonols and flavone luteolin at 410−430 nm after addition of (only) AlCl 3 solution, whereas method 2 investigated the same Al(III)-complexation procedure in the presence of NaNO 2 in alkaline medium and was found specific for rutin, luteolin, and catechins but also phenolic acids at an analytical wavelength of 510 nm. Although these two procedures yielded a different order of flavonoid content for the studied plant extracts (i.e., St. John's wort, green tea, black tea, fruit tea, chamomile, red wine, orange juice, and apple juice), St. John's wort had the highest total flavonoid content with respect to (wrt) procedure 1 and one of the three high ones wrt procedure 2. 45 In addition, rutin was found as the main flavonoid constituent of aqueous and different solvent extracts of St. John's wort. 46 It is also known that compared to the highly colored Al-flavonoid complex, Al(III) easily forms a colorless AlF 6 3− complex with fluoride. 23 With this background, we devised a readily available, nontoxic, economical, and flavonoid-rich aqueous extract (organic solvent-free) from St. John's wort as a natural alternative chromogenic reagent for fluoride when complexed with Al(III), because the color of the Al(III)-flavonoid complex could be bleached by fluoride (due to the formation of the stable hexafluoroaluminate(III) complex) in a concentration-dependent manner, thereby enabling a selective, eco-friendly, and accurate determination of fluoride. 2.2. Apparatus. A Rayleigh VIS-723G visible spectrophotometer and its glass cuvettes (optical thickness, 5 mm) were used for all absorbance measurements. A Precisa XB 220A Analytical Balance was used to weigh all the chemicals, and a Glassco 710 DNAG hot plate with a magnetic stirrer was used to boil the ultrapure water required for the aqueous extract of St. John's wort. A Wisetherm-fuzzy control, wısd HB-48 dry bath was used to find the optimal temperature for the recommended method. , and PO 4 3− ) and cations (K + , NH 4 + , Ag + , Ca 2+ , Mg 2+ , Mn 2+ , Fe 2+ , and Fe 3+ ). The interferences of Fe 2+ , Fe 3+ , and PO 4 3− could be easily eliminated.
First, the maximum amount of Na 2 EDTA that will not dissociate the Al(III)-flavonoid complex in the recommended method was determined. If an EDTA optimization was not carried out, an excess of EDTA could decolorize the Al(III)flavonoid complex as the target probe for fluoride attack. For this purpose, Na 2 EDTA solutions were prepared at initial concentrations of 100, 200, 300, and 400 mg L −1 . Each Na 2 EDTA solution was added instead of fluoride in the proposed method, and ultrapure water was added instead of fluoride for the blank solution. Then, different mass ratios of Na 2 EDTA were tested along with iron ions (having different valencies) in 1:1 iron:fluoride solutions to remove the interference due to Fe 2+ and Fe 3+ ions by masking.
The interference effect of phosphate was eliminated using Ag + ions as a precipitation agent in acidic medium. For this purpose, 0.5 mL of 1000 mg L −1 F − , 0.5 mL of 1000 mg L −1 PO 4 3− , and 0.5 mL of 1000 mg L −1 Ag + were mixed. After the

Method Validation of the Developed Method against the UV−Vis Reference Method for Fluoride
Detection. The recommended method was validated against the slightly modified UV−Vis reference method. 47 The reference and recommended methods were compared at the desired confidence level using the t-and F-statistical tests. The preparation of solution A and solution B and the application of the proposed method are briefly described below: For blank solution, 1.0 mL of solution B + 1.5 mL of ultrapure water (V total = 2.5 mL).
2.8. Statistical Analysis. Descriptive statistical analyses were performed using Excel software (Microsoft Office 2019) for calculating the means and the standard error of the mean. Results were expressed as mean ± standard deviation (SD). Validation of the recommended method for determining the fluoride content against the UV−Vis reference method 47 was made using the statistical tools of the same software.  To find the optimal wavelength, St. John's wort extract (1.0 mL), pH 4.0 acetic acid-acetate buffer solution (1.0 mL, 0.1 M), 250.0 mg L −1 (initial conc.) Al 3+ solution (0.5 mL), and F − at 75.0 mg L −1 initial concentration (0.5 mL) were added to test tubes (V total = 3.0 mL), and for the blank, 0.5 mL of ultrapure water was added instead of F − solution. Then, after the blank and sample solutions were kept for 10.0 min, their absorbances were recorded against water in the wavelength range of 370−550 nm, and the wavelength at which the absorbance difference between the blank and the sample (ΔA) was maximum was 420 nm, as shown in Figure 1a.
To find the optimal Al 3+ concentration, fluoride solutions at 12. (initial conc.) Al 3+ solution + 0.5 mL of F − at initial different concentrations (12.5 or 25.0 mg L −1 ) (V total = 3.0 mL), wait for 10.0 min at RT, and measure absorbance at λ 420 nm against water. For blank solution, 0.5 mL of ultrapure water was added instead of F − . Acetic acid−sodium acetate buffer solution (for pH 4.0 and pH 5.0), potassium hydrogen phthalate-NaOH buffer solution (for pH 6.0), ammonium acetate (for pH 7.0) buffer solution, and borax-HCl buffer solution (for pH 8.0) were used for the optimization. The absorbance differences with two different fluoride concentrations were maximum at pH 4.0, and this pH was chosen as the optimal value as shown in Figure 1c. It may be deduced that as the pH was raised above pH 4, the Al-flavonoid complex became more stable due the deprotonation of phenolic chromogen, thereby making the ligand exchange (i.e., flavonoid displacement with fluoride from the Al(III) coordination sphere) reaction more difficult for fluoride. On the other hand, as the pH was lowered below pH 4, the relative abundance of fluoride decreased because of the formation of weak acid HF and the conditional stability of the Al-flavonoid complex decreased due to the protonation of phenolic chromogen.
To select the optimal time, separate experiments were made for blank solution and 50. For blank solution, 0.5 mL of ultrapure water was added instead of fluoride. After the solutions were added, the blank solutions and samples were incubated separately for up to 25 min at 1 min intervals, and then A 420 nm values were recorded against water. As seen in Figure 1d, the optimal time for the blank solution was 10 min, while for the samples, it was 3 min. It can be said that the interaction of Al(III) with flavonoids in the blank solution had more covalent character than the formation of the AlF 6 3− complex, because the optimal times of the blank solution and samples were different. Since the absorbance readings of the blank solution and samples were recorded against water, the optimal time was chosen as 10 min for the recommended method.
To Since it was observed that the recommended method was independent of temperature as seen in Figure 1e, RT was chosen as the optimal temperature for a more easily applicable method. Actually, Al(III) complexation with fluoride basically depends on electrostatic interactions (i.e., having less covalent character than that with flavonoid), and ionic complexation reactions are known to be less dependent on temperature.

Working Principle of the Recommended Method.
The 3′-4′ dihydroxy group in the B ring, the 3-hydroxy or 5hydroxy groups, and the 4-carbonyl group in the C ring are known as the three possible moieties of flavonoids that are responsible for reacting with metal ions (Figure 2a). 48 As a result of flavonoids forming a coordination compound with metals, their absorption spectra change and a bathochromic (red) shift is observed in the UV−Vis spectrum. According to some authors, the reason for this red shift is a strong charge transfer transition from the flavonoids to the metal center, while others argue that the decrease in the HUMO-LUMO energy levels of the flavonoid molecules is greater than the charge transfer from the ligand to the center. 49 For fluoride determination, the chromogenic agent was formed by providing a red shift in the absorption band due to the formation of the Al(III)-flavonoid complex from the St. John's wort extract solution. Then, by adding fluoride ions to the medium, aluminum ions�capable of forming colored complexes with flavonoids�selectively form a colorless AlF 6 3− complex. Therefore, a blue (hypsochromic) shift in the absorption band of flavonoids is observed (Figure 2b). Taking advantage of this phenomenon, the recommended method is based on the decolorization of the dark yellow color of the chromogenic agent in direct proportion to the amount of fluoride added to the medium. The pH of this competitive ligand exchange reaction for Al(III) was optimized by considering the relative stabilities of Al-flavonoid and Alfluoride complexes.
3.3. Analytical Performance of the Recommended Method for the Determination of Fluoride. It was observed that the decreasing absorbance of ΔA 420 nm ((A o − A i ) 420 nm ) was related to the increasing fluoride concentration when the proposed method was applied. In this recommended method, as the concentration of fluoride solutions increased, the decolorization (from dark yellow to light yellow) of the Al(III)-flavonoid complex was observed (Figure 3). All the absorbance measurements were recorded against water at 420 nm wavelength. The linear calibration equation was obtained with the data of ΔA 420 nm against the fluoride concentration (eq 1).
Linear calibration equation for fluoride: (1) where C F − is the final concentration of fluoride (in millimoles per liter).
The linear concentration range was from 0.11 to 1.32 mM (2.0 to 25.0 mg L −1 ), covering an order of magnitude for C F − . In addition, the molar absorptivity (ε), limit of detection (LOD), and limit of quantification (LOQ) values were 7.67 × 10 2 ± 2.2 × 10 1 L mol −1 cm −1 , 0.026 mM (0.5 mg L −1 ), and 0.079 mM (1.5 mg L −1 ), respectively. The limit of detection was found in millimole per liter units (LOD = 3σ bl /m, with σ bl denoting the standard deviation of a blank and m showing the slope of the calibration line). The coefficients of variation (CVs) of intra-and inter-assay for fluoride were 2.17 and 2.59%, respectively (N = 5), showing that the recommended method has good precision. The LOD value is below the permissible limit of fluoride in water (0.079 mM, 1.5 mg L −1 ) by WHO 50 and also enforceable (0.22 mM, 4.0 mg L −1 ) and non-enforceable (secondary, 0.11 mM (2.0 mg L −1 )) limits by EPA. 51,52 In addition, the recommended method and previously published fluoride determination methods were compared with respect to certain parameters such as solution medium, linear concentration range, LOD values, and real sample application in Table 1.  Table 2, and the fluoride recovery (%) values were found between 88.2 and 108.0% (Figure 4).

Investigation of Interference Effect of Common
The initial concentration of Na 2 EDTA, forming a complex with the interfering iron ions (Fe 2+ and Fe 3+ ) but not interacting with aluminum in the Al(III)-flavonoid complex, was investigated. For this purpose, a maximal initial concentration of 300.0 mg L −1 Na 2 EDTA was added instead of fluoride, and no change was observed in the absorbance of the blank solution. The interference effect of Fe 2+ and Fe 3+ was easily eliminated by using Na 2 EDTA as a masking agent, which was added at different mass ratios for each valency of iron [metal−EDTA ratio: 1:3 (w/w) for Fe 2+ and 1:6 (w/w) for Fe 3+ ] to 1:1 iron:fluoride solutions before applying the recommended method. Since ferric ions had a higher affinity for fluoride than ferrous ions in accordance with crystal field theory, they required a higher concentration of the chelating agent (EDTA) for sufficient masking. During this procedure, the Al(III)-flavonoid complex was not affected because Al(III) was already bound in a stable complex, and Al(III) chelation with EDTA needs temperature and time (i.e., Al(III) has slow kinetics due to its noble gas electronic configuration).
Elimination of the interference effect of phosphate is based on the principle of removing it from solution by precipitation with silver ions in acidic medium (details are given in Section 2.5). The proposed method was applied to solutions having    The proposed method was applied to mineral water with standard additions, with a final concentration range of 4.447− 16.947 mg L −1 (details are given in Section 2.6). Fluoride recoveries (%) and RSD % were found to be between 99.8 and 102.9% and between 0.60 and 4.44%, respectively, as shown in Table 3.
According to the literature, 60 artificial wastewater was prepared with a mixture of initial concentrations of 100.0 mg L −1 B (H 3 BO 3 was used as a boron source) and 300.0 mg L −1 F − . Concrete sludge is an industrial waste slurry containing hydrated cement, aggregates, and water, and its wastewater may contain both boron and fluoride. 60 The proposed method was applied to artificial wastewater with standard additions at a final concentration range of 4.166−16.666 mg L −1 (details are given in Section 2.6). Fluoride recoveries (%) and RSD % were found to be between 93.6 and 101.6% and between 0.21 and 1.39%, respectively, as shown in Table 3. After preparing fluoride solutions to have an initial concentration of 75 mg L −1 in the artificial wastewater from five different mixtures with initial concentrations of 300 mg L −1 F − and 100 mg L −1 B solutions (H 3 BO 3 as the boron source), absorbance readings were recorded for the recommended method and the reference method (final conc. of F − was 12.5 mg L −1 when both methods were applied). The mean of the absorbance readings of three consecutive samples was used for each calculation (N = 3). The results showed no significant difference in precision and accuracy between the two methods. The recommended method was validated against the UV−Vis reference method at the 95% confidence level using Student's t-and F-tests. Statistical parameters of the recommended method and the UV−Vis reference method are depicted in Table 4.

CONCLUSIONS
In this study, our main aim was to develop a simple and inexpensive fluoride determination method based on the principles of green analytical chemistry, free from toxic solvents. This method was eco-friendly without complicated steps and utilized a natural extract in a green chromogenic method that meets today's requirements. For this purpose, St. John's wort was chosen as a natural sensing reagent for fluoride determination by utilizing the Al(III)-flavonoid metal complex formation due to the strong charge transfer interaction of flavonoids with metal ions and/or the decrease in HUMO-LUMO energy levels of flavonoids, which is more effective than ligand-to-metal charge transfer. It is clear that the LOD value (0.5 mg L −1 ) of the recommended method meets the required critical values of WHO (1.5 mg L −1 ) and EPA (4 mg L −1 for the non-enforceable standard and 2 mg L −1 for the secondary standard) for the fluoride content of water samples. The possible interferences of ionic species commonly found in water were investigated for establishing the selectivity of the  recommended method for the analyte. While Na 2 EDTA was used to selectively remove the interference effects of Fe 2+ and Fe 3+ , the precipitation method was applied in acidic medium to eliminate the interference of PO 4 3− using Ag + ions as a precipitation agent. The recommended method was applied to water samples (mineral water and artificial wastewater from concrete sludge) and also validated against a reference UV−Vis reference method at the 95% confidence level.  S 2 = ((n 1 − 1)s 1 2 + (n 2 − 1)s 2 2 )/(n 1 + n 2 − 2) and t = (a ̅ 1 − a ̅ 2 )/(S(1/n 1 + 1/n 2 ) 1/2 ), where S is the pooled standard deviation, s 1 and s 2 are the standard deviations of the two populations with sample sizes of n 1 and n 2 , and a ̅ 1 and a ̅ 2 are sample means (t has (n 1 + n 2 − 2) degrees of freedom); here, n 1 = n 2 = 5. b Statistical comparison made on paired data produced with the proposed and reference methods; results given only on the row of the reference method.